A class of calcium-activated potassium channels termed BK(Ca) channels are enriched at presynaptic neurotransmitter release sites within the brain, and are believed to control both the amount and timing of neurotransmitter release. BK(Ca) channels are encoded by the Slo gene. Recent evidence suggests that a related gene, Slack, also contributes to BK(Ca)-like channels in neurons, and that some BK(Ca) channels may be comprised of heteromultimers of Slo and Slack channels subunits. The experiments in this proposal will characterize the molecular, biophysical and structural properties of BK(Ca) channels in both the somata and presynaptic terminals of native neurons, and in transfected cells. The role of the Slo and Slack channel subunits in normal synaptic transmission, and the mechanisms of their response to hypoxia, will be determined by patch clamp measurements coupled with genetic knockout approaches. The structure and function of the large carboxy-terminal regulatory region of the Slo channel subunit with its calcium-binding sites, and its interaction with other proteins, including the Slack subunit, will be determined by biophysical and biochemical measurements and by X-ray crystallographic methods. Finally the structure of intact BK(Ca) channels will be determined by cryo-electronmicroscopy. Knowledge of the molecular properties and regulation of BK(Ca) channels in normal and hypoxic neurons, together with structural determinations of their interactions with both drugs and natural ligands, will be key in the development of more effective treatments for disorders such as epilepsy and stroke.